Genome-guided discovery of diverse natural products from Burkholderia sp.

Burkholderia species have emerged as a new source of diverse natural products. This mini-review covers all of the natural products discovered in recent years from Burkholderia sp. by genome-guided approaches—these refer to the use of bacterial genome sequence as an entry point for in silico structural prediction, wet lab experimental design, and execution. While reliable structural prediction based on cryptic biosynthetic gene cluster sequence was not always possible due to noncanonical domains and/or module organization of a deduced biosynthetic pathway, a molecular genetic method was often employed to detect or alter the expression level of the gene cluster to achieve an observable phenotype, which facilitated downstream natural product purification and identification. Those examples of natural product discovery from Burkholderia sp. provide practical guidance for future exploration of Gram-negative bacteria as a new source of natural products.     

A peroxisomal thioesterase plays auxiliary roles in plant β‐oxidative benzoic acid metabolism

Peroxisomal β‐oxidative degradation of compounds is a common metabolic process in eukaryotes. Reported benzoyl‐coenzyme A (BA‐CoA) thioesterase activity in peroxisomes from petunia flowers suggests that, like mammals and fungi, plants contain auxiliary enzymes mediating β‐oxidation. Here we report the identification of Petunia hybrida thioesterase 1 (PhTE1), which catalyzes the hydrolysis of aromatic acyl‐CoAs to their corresponding acids in peroxisomes. PhTE1 expression is spatially, developmentally and temporally regulated and exhibits a similar pattern to known benzenoid metabolic genes. PhTE1 activity is inhibited by free coenzyme A (CoA), indicating that PhTE1 is regulated by the peroxisomal CoA pool. PhTE1 downregulation in petunia flowers led to accumulation of BA‐CoA with increased production of benzylbenzoate and phenylethylbenzoate, two compounds which rely on the presence of BA‐CoA precursor in the cytoplasm, suggesting that acyl‐CoAs can be exported from peroxisomes. Furthermore, PhTE1 downregulation resulted in increased pools of cytoplasmic phenylpropanoid pathway intermediates, volatile phenylpropenes, lignin and anthocyanins. These results indicate that PhTE1 influences (i) intraperoxisomal acyl‐CoA/CoA levels needed to carry out β‐oxidation, (ii) efflux of β‐oxidative products, acyl‐CoAs and free acids, from peroxisomes, and (iii) flux distribution within the benzenoid/phenylpropanoid metabolic network. Thus, this demonstrates that plant thioesterases play multiple auxiliary roles in peroxisomal β‐oxidative metabolism.     

Methylmalonyl coenzyme A selectivity of cloned and expressed acyltransferase and beta-ketoacyl synthase domains of mycocerosic acid synthase from Mycobacterium bovis BCG.

Methyl-branched fatty acids and polyketides occur in a variety of living organisms. Previous studies have established that multifunctional enzymes use methylmalonyl coenzyme A (CoA) as the substrate to generate methyl-branched products such as mycocerosic acids and polyketides. However, we do not know which of the component activities show selectivity for methylmalonyl-CoA in any biological system. A comparison of homologies of the domains of the multifunctional synthases that selectively use malonyl-CoA or methylmalonyl-CoA suggested that the acyltransferase (AT) and beta-ketoacyl synthase (KS) domains might be responsible for the substrate selectivity. To test this hypothesis, we expressed the AT and KS domains of the mycocerosic acid synthase (MAS) gene from Mycobacterium bovis BCG in Escherichia coli and examined whether they confer to synthases that normally do not use methylmalonyl-CoA the ability to incorporate methylmalonyl-CoA into fatty acids. Both the AT and the KS domains of MAS showed selectivity for methylmalonyl-CoA over malonyl-CoA. Acyl carrier protein (ACP)-dependent elongation of the n-C12 acyl primer mainly by one methylmalonyl-CoA unit was catalyzed by an E. coli fatty acid synthase preparation only in the presence of the expressed MAS domains. An ACP-dependent elongation of the n-C20 acyl primer by one methylmalonyl-CoA extender unit was catalyzed by fatty acid synthase from Mycobacterium smegmatis only in the presence of the expressed MAS domains. These results show methylmalonyl-CoA selectivity for the AT and KS domains of MAS. These domains may be useful in producing novel polyketides by genetic engineering.     

New enzymes for peptide biosynthesis in microorganisms

Peptides, biologically occurring oligomers of amino acids linked by amide bonds, are essential for living organisms. Many peptides isolated as natural products have biological functions such as antimicrobial, antivirus and insecticidal activities. Peptides often possess structural features or modifications not found in proteins, including the presence of nonproteinogenic amino acids, macrocyclic ring formation, heterocyclization, N-methylation and decoration by sugars or acyl groups. Nature employs various strategies to increase the structural diversity of peptides. Enzymes that modify peptides to yield mature natural products are of great interest for discovering new enzyme chemistry and are important for medicinal chemistry applications. We have discovered novel peptide modifying enzymes and have identified: (i) a new class of amide bond forming-enzymes; (ii) a pathway to biosynthesize a carbonylmethylene-containing pseudodipeptide structure; and (iii) two distinct peptide epimerases. In this review, an overview of our findings on peptide modifying enzymes is presented.     

Biophysical and biochemical mechanisms by which dietary N-3 polyunsaturated fatty acids from fish oil disrupt membrane lipid rafts

N-3 polyunsaturated fatty acids (PUFAs) from fish oil exert their functional effects by targeting multiple mechanisms. One mechanism to emerge in the past decade is the ability of n-3 PUFA acyl chains to perturb the molecular organization of plasma membrane sphingolipid/cholesterol-enriched lipid raft domains. These domains are nanometer-scale assemblies that coalesce to compartmentalize select proteins for optimal function. Here we review recent evidence on how n-3 PUFAs modify lipid rafts from biophysical and biochemical experiments from several different model systems. A central theme emerges from these studies. N-3 PUFA acyl chains display tremendous conformational flexibility and a low affinity for cholesterol and saturated acyl chains. This unique flexibility of n-3 PUFA acyl chains impacts the organization of inner and outer leaflet lipid rafts by disrupting acyl chain packing and molecular order within rafts. Ultimately, the disruption in raft organization has consequences for protein clustering and thereby signaling. Overall, elucidating the complex mechanisms by which n-3 PUFA acyl chains reorganize membrane architecture will enhance the translation of these fatty acids into the clinic for treating several diseases.     

植物丝氨酸羧肽酶样酰基转移酶的结构、功能和应用

植物丝氨酸羧肽酶样酰基转移酶与丝氨酸羧肽酶相比具有相似的结构特点和很高的同源性,能够催化酰基葡萄糖酯的酰基转移反应,参与植物次生代谢产物的酰基化修饰,丰富天然产物结构多样性,改善化合物水溶性、稳定性等理化性质。本文重点介绍植物来源丝氨酸羧肽酶样酰基转移酶家族的结构特点、催化机制、功能鉴定及其生物催化应用等方面的研究进展,为促进此类酰基转移酶基因的功能表征和利用合成生物技术合成活性次生代谢产物提供参考。     

CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function

The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve", while the reaction products of amino acids smaller than the cognate substrate will go through a "fine sieve" and be hydrolyzed. This "double-sieve" mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain). This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNA(Leu) by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA(Leu) made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function.     

Efforts towards the design of 'teflon' proteins: in vivo translation with trifluorinated leucine and methionine analogues

In vivo incorporation of monofluorinated noncanonical amino acids into recombinant proteins has been well-established for decades. Proteins fluorinated in this way proved to be useful tools for many practical applications. In contrast, trifluorinated amino acids have been incorporated in only a few peptides and relatively small proteins by using expression systems in living cells. A novel class of proteins with a fluorous core can be envisaged only if full replacement of the core-building hydrophobic and aliphatic amino acids such as leucine or methionine with the related analogues trifluoromethionine and trifluoroleucine would be feasible. However, our systematic efforts to introduce these amino acids in larger proteins (over 10 Da) that contain different structural motifs clearly show that only partial substitutions are possible. The reasons are high toxicity of these substances and difficulties to accommodate them into the compact cores of natural proteins without adverse effects on their structural integrity. Therefore, engineering of such three dimensional 'Teflon'-like structures would require, besides an expansion of the amino acid repertoire of the genetic code, a de novo protein design as well.     

色氨酸, 天然产物生物合成中的重要结构单元

氨基酸作为生物体内组成生命物质的小分子化合物, 在天然产物生物合成中扮演了非常重要的作用。色氨酸含有一个独特的吲哚环, 相对复杂的吲哚环平面结构使得色氨酸相比其他氨基酸具有更多的修饰空间。在微生物天然产物生物合成研究中, 色氨酸及其衍生物经常作为组成模块参与到天然产物的生物合成中, 本文概述了色氨酸几种不同的生物修饰方式, 包括烷基化修饰、卤化修饰、羟基化修饰、以及吲哚环的开环重排反应等。分析并总结色氨酸在天然产物生物合成中的作用可以增加我们对天然产物结构多样性的认识和推动天然产物生物合成机制的研究。     

Acyl chain specificity of the acyltransferases LpxA and LpxD and substrate availability contribute to lipid A fatty acid heterogeneity in Porphyromonas gingivalis

Porphyromonas gingivalis lipid A is heterogeneous with regard to the number, type, and placement of fatty acids. Analysis of lipid A by matrix-assisted laser desorption ionization-time of flight mass spectrometry reveals clusters of peaks differing by 14 mass units indicative of an altered distribution of the fatty acids generating different lipid A structures. To examine whether the transfer of hydroxy fatty acids with different chain lengths could account for the clustering of lipid A structures, P. gingivalis lpxA (lpxA(Pg)) and lpxD(Pg) were cloned and expressed in Escherichia coli strains in which the homologous gene was mutated. Lipid A from strains expressing either of the P. gingivalis transferases was found to contain 16-carbon hydroxy fatty acids in addition to the normal E. coli 14-carbon hydroxy fatty acids, demonstrating that these acyltransferases display a relaxed acyl chain length specificity. Both LpxA and LpxD, from either E. coli or P. gingivalis, were also able to incorporate odd-chain fatty acids into lipid A when grown in the presence of 1% propionic acid. This indicates that E. coli lipid A acyltransferases do not have an absolute specificity for 14-carbon hydroxy fatty acids but can transfer fatty acids differing by one carbon unit if the fatty acid substrates are available. We conclude that the relaxed specificity of the P. gingivalis lipid A acyltransferases and the substrate availability account for the lipid A structural clusters that differ by 14 mass units observed in P. gingivalis lipopolysaccharide preparations.     

Nucleotide sequence of yeast gene CP A1 encoding the small subunit of arginine-pathway carbamoyl-phosphate synthetase. Homology of the deduced amino acid sequence to other glutamine amidotransferases

A yeast DNA fragment carrying the gene CP A1 encoding the small subunit of the arginine pathway carbamoyl-phosphate synthetase has been sequenced. Only one continuous coding sequence on this fragment was long enough to account for the presumed molecular mass of CP A1 protein product. It codes for a polypeptide of 411 amino acids having a relative molecular mass, Mr, of 45 358 and showing extensive homology with the product of carA, the homologous Escherichia coli gene. CP A1 and carA products are glutamine amidotransferases which bind glutamine and transfer its amide group to the large subunits where it is used for the synthesis of carbamoyl-phosphate. A comparison of the amino acid sequences of CP A1 polypeptide with the glutamine amidotransferase domains of anthranilate and p-amino-benzoate synthetases from various sources has revealed the presence in each of these sequences of three highly conserved regions of 8, 11 and 6 amino acids respectively. The 11-residue oligopeptide contains a cysteine which is considered as the active-site residue involved in the binding of glutamine. The distances (number of amino acid residues) which separate these homology regions are accurately conserved in these various enzymes. These observations provide support for the hypothesis that these synthetases have arisen by the combination of a common ancestral glutamine amidotransferase subunit with distinct ammonia-dependent synthetases. Little homology was detected with the amide transfer domain of glutamine phosphoribosyldiphosphate amidotransferase which may be the result of a convergent evolutionary process. The flanking regions of gene CP A1 have been sequenced, 803 base pairs being determined on the 5' side and 382 on the 3' side. Several features of the 5'-upstream region of CP A1 potentially related to the control of its expression have been noticed including the presence of two copies of the consensus sequence d(T-G-A-C-T-C) previously identified in several genes subject to the general control of amino acid biosynthesis.     

The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid

The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.     

Genetic analysis of polyketide synthase and peptide synthetase genes in cyanobacteria as a mining tool for secondary metabolites

Molecular screening using degenerate PCR to determine the presence of secondary metabolite genes in cyanobacteria was performed. This revealed 18 NRPS and 19 PKS genes in the 21 new cyanobacterial strains examined, representing three families of cyanobacteria (Nostocales, Chroococales and Oscillatoriales). A BLAST analysis shows that these genes have similarities to known cyanobacterial natural products. Analysis of the NRPS adenylation domain indicates the presence of novel features previously ascribed to both proteobacteria and cyanobacteria. Furthermore, binding-pocket predictions reveal diversity in the amino acids used during the biosynthesis of compounds. A similar analysis of the PKS ketosynthase domain shows significant structural diversity and their presence in both mixed modules with NRPS domains and individually as part of a PKS module. We have been able to classify the NRPS genes on the basis of their binding-pockets. Further, we show how this data can be used to begin to link structure to function by an analysis of the compounds Scyptolin A and Hofmannolin from Scytonema sp. PCC 7110.     

Specificity of TRAF3 in its negative regulation of the noncanonical NF-kappa B pathway

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are critical signaling adaptors downstream of many receptors in the TNF receptor and interleukin-1 receptor/Toll-like receptor superfamilies. Whereas TRAF2, 5, and 6 are activators of the canonical NF-kappaB signaling pathway, TRAF3 is an inhibitor of the noncanonical NF-kappaB pathway. The contribution of the different domains in TRAFs to their respective functions remains unclear. To elucidate the structural and functional specificities of TRAF3, we reconstituted TRAF3-deficient cells with a series of TRAF3 mutants and assessed their abilities to restore TRAF3-mediated inhibition of the noncanonical NF-kappaB pathway as measured by NF-kappaB-inducing kinase (NIK) protein levels and processing of p100 to p52. We found that a structurally intact RING finger domain of TRAF3 is required for inhibition of the noncanonical NF-kappaB pathway. In addition, the three N-terminal domains, but not the C-terminal TRAF domain, of the highly homologous TRAF5 can functionally replace the corresponding domains of TRAF3 in suppression of the noncanonical NF-kappaB pathway. This functional specificity correlates with the specific binding of TRAF3, but not TRAF5, to the previously reported TRAF3 binding motif in NIK. Our studies suggest that both the RING finger domain activity and the specific binding of the TRAF domain to NIK are two critical components of TRAF3 suppression of NIK protein levels and the processing of p100 to p52.     

Intra-chain 3D segment swapping spawns the evolution of new multidomain protein architectures

Multidomain proteins form in evolution through the concatenation of domains, but structural domains may comprise multiple segments of the chain. In this work, we demonstrate that new multidomain architectures can evolve by an apparent three-dimensional swap of segments between structurally similar domains within a single-chain monomer. By a comprehensive structural search of the current Protein Data Bank (PDB), we identified 32 well-defined segment-swapped proteins (SSPs) belonging to 18 structural families. Nearly 13% of all multidomain proteins in the PDB may have a segment-swapped evolutionary precursor as estimated by more permissive searching criteria. The formation of SSPs can be explained by two principal evolutionary mechanisms: (i) domain swapping and fusion (DSF) and (ii) circular permutation (CP). By large-scale comparative analyses using structural alignment and hidden Markov model methods, it was found that the majority of SSPs have evolved via the DSF mechanism, and a much smaller fraction, via CP. Functional analyses further revealed that segment swapping, which results in two linkers connecting the domains, may impart directed flexibility to multidomain proteins and contributes to the development of new functions. Thus, inter-domain segment swapping represents a novel general mechanism by which new protein folds and multidomain architectures arise in evolution, and SSPs have structural and functional properties that make them worth defining as a separate group.     

Characterization of type II thioesterases involved in natamycin biosynthesis in Streptomyces chattanoogensis L10

The known functions of type II thioesterases (TEIIs) in type I polyketide synthases (PKSs) include selecting of starter acyl units, removal of aberrant extender acyl units, releasing of final products, and dehydration of polyketide intermediates. In this study, we characterized two TEIIs (ScnI and PKSIaTEII) from Streptomyces chattanoogensis L10. Deletion of scnI in S. chattanoogensis L10 decreased the natamycin production by about 43%. Both ScnI and PKSIaTEII could remove acyl units from the acyl carrier proteins (ACPs) involved in the natamycin biosynthesis. Our results show that the TEII could play important roles in both the initiation step and the elongation steps of a polyketide biosynthesis; the intracellular TEIIs involved in different biosynthetic pathways could complement each other.